Methods and Compositions for the Treatment of Cellulosic Biomass and Products Produced Thereby

ABSTRACT

A two-step method for activating a cellulosic feedstock is described. The feedstock is subjected to a first high temperature activation step at a temperature greater than 190° C. and a second activation step at a lower temperature under alkali conditions. Also described are methods and compositions for the enzymatic hydrolysis of activated cellulose using one or more cellulase enzymes, a surfactant and polyaspartic acid. Also described are products of the methods.

CROSS REFERENCE TO PRIOR APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/565,069, filed Oct. 6, 2017, which is a National Stage applicationunder 35 U.S.C. § 371 of International Application No.PCT/CA2016/050402, filed Apr. 8, 2016, which claims priority under theParis Convention to U.S. Application No. 62/246,271, filed Oct. 26,2015, and to U.S. Application No. 62/145,785, filed Apr. 10, 2015, theentire contents of which are incorporated herein by reference.

FIELD

This application relates to methods for treating cellulosic biomass toproduce cellulosic sugars. In one aspect, methods for activatingcellulosic feedstock and/or enzymatic hydrolysis to produce glucose areprovided. Activated cellulosic feedstock and products produced fromenzymatic hydrolysis of the activated cellulosic feedstock are alsoprovided.

INTRODUCTION

The production of sugars such as glucose from cellulosic biomass hasbeen the focus of considerable research and development. However, thehigh cost and low conversion rate of many processes has limited thewidespread adoption of cellulosic sugar technology.

A number of different methods for converting cellulosic biomass intosugars are known in the art. These generally include a pretreatment stepwherein cellulosic biomass is physically and/or chemically altered toopen up the structure of the polymeric sugars contained in cellulosicbiomass and an enzymatic or chemical hydrolysis step wherein thepolymeric sugars are broken down into monomeric sugars.

While high yields of glucose (>90%) based on cellulose have beenreported, these yields are normally achieved at low concentrations ofglucose, typically 2-5%. Methods that result in both a high yield and ahigh concentration of glucose are difficult to achieve because thepresence of glucose typically reduces the activity of cellulase enzymeseven at high enzyme loadings. Cellulase enzyme activity decreases overtime, necessitating the addition of fresh enzyme to enzymatic hydrolysisreactions to maintain yield. However, the high cost of cellulase enzymescan become prohibitive. Cellulase enzymes may also bind withrecalcitrant cellulose and/or lignin and become unavailable for thefurther hydrolysis of cellulose to glucose. This non-productive bindingalso hinders the recycling of enzymes, which is desirable to reduceenzyme usage and lower operating costs.

Various different processes have been developed for pre-treating thecellulosic feedstock and various enzymatic hydrolysis processes havebeen developed to convert the treated cellulosic feedstock to sugars.For example, Parekh (PCT Publication No. WO2014/026154) describe atwo-stage pre-treatment process for lignocellulosic biomass largelyunder acidic conditions. Schiffino et al. (US Publication No.2011/0250645) describe methods for improving the release of monomericsugars from alkaline treated biomass. Liu et al. (US Publication No.2011/0300586) describe a two-stage pretreatment process forlignocellulosic biomass with the objective of reducing the crystallinityof the cellulose and to dissociate the hemicellulose-cellulose complex.Embodiments include a low severity steam treatment or autohydrolysisfollowed by hydrolysis with dilute acid or hot water.

SUMMARY

This summary is intended to introduce the reader to the more detaileddescription that follows and not to limit or define any claimed or asyet unclaimed invention. One or more inventions may reside in anycombination or sub-combination of the elements or process stepsdisclosed in any part of this document including its claims and FIGURE.

According to one broad aspect, there is provided a method for activatinga cellulosic feedstock in order to increase the chemical and/orenzymatic reactivity of cellulose in the feedstock. Activated cellulosemay then be converted into cellulosic sugars such as by subjecting theactivated cellulose to enzymatic hydrolysis.

In accordance with this aspect, the cellulosic feedstock may besubjected to a first high temperature activation step followed by asecond activation step at a lower temperature under alkali conditions.In accordance with this embodiment, the method may comprise subjectingthe feedstock to a first activation step wherein the feedstock istreated at a temperature greater than 190° C. and a pressure greaterthan 200 psig to produce a first activated cellulose stream comprisingcellulose II and insoluble solids. The insoluble solids may includecomponents of the feedstock other than cellulose such as lignin.Subsequently, the first activated cellulose stream may be subjected to asecond activation step wherein the first activated cellulose stream istreated with an alkali at a lower temperature than the first activationstep to produce a second activated cellulose stream comprising celluloseIV. Preferably, the first activation step is conducted in the presenceof water.

Without being limited by theory, it is believed that the firstactivation step alters the crystalline state of cellulose in thecellulosic feedstock to produce a first activated cellulose stream witha higher proportion of cellulose II relative to the amount of celluloseII in the cellulosic feedstock. The second activation step is believedto further alter the crystalline state of cellulose in the firstactivated cellulose stream to produce a second activated cellulosestream with a higher proportion of cellulose IV relative to the amountof cellulose IV in the first activated cellulose stream. In oneembodiment, the two-step activation method described herein produces amixture of cellulose II, hydrated cellulose II and alkali-cellulose IV.Optionally, the cellulosic material may be treated, e.g., washed and/orfiltered, after one or each activation step in order to remove solublenon-cellulosic components.

The method for activating cellulose described herein has also beendetermined to result in activated cellulose with an increase in thelevel of glucan and/or a decrease in the level of non-cellulosiccomponents of the cellulosic feedstock such as lignin. For example, inone embodiment the methods described herein produce activated cellulosewith at least 60%, at least 70% or at least 75% glucan. In oneembodiment, the methods described herein produce activated cellulosewith less than 25%, less than 20% or less than 15% lignin.

According to another broad aspect, methods and compositions are providedfor stabilizing enzymes during enzymatic hydrolysis, maintainingenzymatic activity and/or obtaining an enzyme recycling stream.

According to another broad aspect, an enzymatic hydrolysis mix suitablefor use in the enzymatic hydrolysis of cellulose is provided. In apreferred embodiment, the enzymatic hydrolysis mix is contacted withactivated cellulose produced according the methods described herein.

In accordance with these aspects, there is provided one or morecellulase enzymes in combination with a surfactant and/or a dispersantfor the enzymatic hydrolysis of cellulose. Without being limited bytheory, it is believed that the cellulase enzymes form a complex withthe surfactant and/or dispersant that may stabilize the enzymes, helpmaintain enzyme activity, prevent enzyme degradation and/or facilitaterecovery of the enzymes following enzymatic hydrolysis. It is alsobelieved that the presence of a dispersant such as an oligopeptide helpsprevent the non-productive binding of cellulase enzymes by interactingwith lignin and/or other non-cellulose components. In a preferredembodiment, the surfactant is a non-ionic surfactant such as apolysorbate surfactant. In another preferred embodiment, the surfactantis a blend of surfactants, such as Tween™, an alkoxylated glyceride andnonyl phenol. In one embodiment, the dispersant is a non-enzymaticoligopeptide, optionally a polyamino acid, optionally a polyamino acidwith a molecular weight of between 500 and 10,000, between 1000 and 5000or between 3500 and 4500. In a preferred embodiment, the polyamino acidis polyaspartic acid.

The methods and compositions described herein offer a number ofadvantages with respect to the activation of cellulose and/or theproduction of cellulosic sugars, which may be obtained from some of theembodiments. For example, in some embodiments, use of the methods andcompositions described herein may result in a glucose-rich sugar streamwith greater than about 12% glucose, greater than about 14% glucose,greater than about 16% glucose or greater than about 18% glucose.Further, in these or other embodiments, the methods and compositionsdescribed herein may result in a high yield of monomeric sugars. Forexample, in some embodiments, the methods and compositions describedherein may result in a yield of glucose that is greater than about 70%,greater than about 80%, greater than about 85%, greater than about 90%or greater than about 95% of a theoretical yield of glucose. Thetheoretical yield of glucose in an enzymatic hydrolysis reaction may bedetermined based on the glucan content of the activated cellulosicmaterial subjected to enzymatic hydrolysis. In some preferredembodiments, the methods and compositions described herein may result ina glucose rich sugar stream with both a high yield and a highconcentration of glucose. For example, in one embodiment theglucose-rich sugar stream has greater than about 12% glucose and a yieldof greater than 70%, or greater than 14% glucose and a yield of greaterthan 80%, or greater than 16% glucose and a yield greater than 90%.

According to another broad aspect, methods are provided for theenzymatic hydrolysis of activated cellulose to produce cellulosic sugarssuch as glucose. In accordance with this embodiment, the enzymatichydrolysis may be conducted on as a batch process or a continuousprocess. The enzymatic hydrolysis may be conducted using the enzymatichydrolysis mix and/or the activated cellulose as disclosed herein.

In accordance with another aspect, methods are provided for treating aglucose rich sugar stream to remove enzymes used for enzymatichydrolysis. Removing and/or recycling the enzymes used for enzymatichydrolysis may reduce the amount of enzyme needed for enzymatichydrolysis and therefore the costs associated with producing cellulosicsugars. For example, the methods and compositions described herein maybe used to recover at least 60%, at least 70%, at least 80%, or at least85% of the cellulase enzyme activity in an enzyme recycle streamfollowing enzymatic hydrolysis. The enzyme recycling stream may berecycled to continue treating activated cellulose and/or may be used totreat fresh activated cellulose, such as a second activated cellulosestream as described herein. In some embodiments, the glucose rich sugarstream is subjected to multiple enzyme removal treatments, either thesame enzyme removal treatment repeated more than once or differentenzyme removal treatments.

In accordance with another aspect, there is provided a glucose-richsugar stream produced by a method as described herein. In oneembodiment, the sugar stream comprises greater than 12%, greater than14%, greater than 16% or greater than 18% glucose. In one embodiment,the sugar stream comprises polyaspartic acid. In some embodiments, thepolyaspartic acid is present at a concentration between 1 ppb and 10000ppm.

In accordance with another aspect, there is provided a method forproducing a glucose-rich sugar stream comprising (a) providing activatedcellulose comprising a mix of cellulose II, hydrated cellulose II andalkali-cellulose IV; and subjecting the activated cellulose to enzymatichydrolysis with one or more cellulase enzymes, a surfactant and adispersant to produce the glucose-rich sugar stream. Optionally, theactivated cellulose is produced using a method a described herein.

In accordance with another aspect, there is provided a glucose-richsugar stream further comprising a non-glucose sugar, wherein thenon-glucose sugar is one or more of xylose, xylo-oligosaccharide andxylan. In one embodiment the non-glucose sugars comprise about 3-8%,about 4-7% or about 5-6% of the dry matter of the composition. In aparticular embodiment the glucose-rich sugar stream comprises about 5%non-glucose sugar.

In accordance with yet a further aspect of the invention there isprovided a fructose-rich sugar stream prepared by conversion of theglucose in the glucose-rich sugar stream of the invention to fructose.The fructose-rich sugar stream further comprising a non-fructose sugar,wherein the non-fructose sugar is one or more of xylose,xylo-oligosaccharide and xylan. In one embodiment the non-fructosesugars comprise about 1-8%, about 2-7% or about 3-6% of the dry matterof the composition. In a particular embodiment the fructose-rich sugarstream comprises about 5% non-glucose sugar.

In accordance with another aspect of the invention there is provided alower glycemic index glucose syrup or lower glycemic index fructosesyrup wherein the glucose or fructose syrup comprises about 1-8%, about2-7% or about 3-6% of one or more of xylose, xylo-oligosaccharide andxylan, and wherein the glycemic index is lower than the glycemic indexof conventional glucose or fructose syrup produced in a conventionalmanner.

Other features and advantages of the present disclosure will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating embodiments of the disclosure are given by wayof illustration only, since various changes and modifications within thespirit and scope of the disclosure will become apparent to those skilledin the art from this detailed description. In particular, it will beappreciated that any method may use all of the aspects disclosed hereinor any particular combination or sub-combination of the aspects.

DRAWINGS

The drawings included herewith are for illustrating various examples ofmethods, and compositions of the teaching of the present specificationand are not intended to limit the scope of what is taught in any way.

The disclosure will now be described in relation to the drawing inwhich:

FIG. 1 is a schematic flow chart of the method according to a preferredembodiment that includes a two-step activation of a cellulosicfeedstock, enzymatic hydrolysis of the activated feedstock and an enzymeremoval treatment to obtain an enzyme recycle stream and a glucose-richand enzyme-poor sugar stream.

DESCRIPTION OF VARIOUS EMBODIMENTS

Various methods and compositions will be subsequently described toprovide an example of an embodiment of each claimed invention. Noembodiment subsequently described limits any claimed invention and anyclaimed invention may cover methods and compositions that differ fromthose subsequently described. The claimed inventions are not limited tomethods and compositions having all of the features of any one methodand composition subsequently described or to features common to multipleor all of the methods and compositions described below. It is possiblethat a method or composition subsequently described is not an embodimentof any claimed invention. Any invention disclosed in an method orcomposition subsequently described that is not claimed in this documentmay be the subject matter of another protective instrument, for example,a continuing patent application, and the applicants, inventors or ownersdo not intend to abandon, disclaim or dedicate to the public any suchinvention by its disclosure in this document.

Described herein are various methods and compositions useful for thetreatment of cellulosic biomass to produce cellulosic sugars. In oneembodiment, there is provided a method for activating a cellulosicfeedstock to produce activated cellulose. It has been determined thatsubjecting a cellulosic feedstock to a first activation step at a hightemperature and pressure followed by a second activation step with analkali at a lower temperature than the first activation step producesactivated cellulose with chemical and/or physical properties that areadvantageous for the hydrolysis of cellulose into monomeric sugars.

The methods disclosed herein utilize a cellulosic feedstock 10.Cellulosic feedstock 10 may be any feedstock known in the cellulosicsugar art. For example, the cellulosic feedstock may comprise one ormore of straw, corn stover, bagasse, hardwoods, softwoods, energy cropsand the like.

The raw agricultural material which is provided to a plant may betreated to remove rocks, soil and other material present in the rawagricultural material and to reduce the size of the raw agricultural orforest based material that is fed to the process, such as bycomminution, grinding, milling or otherwise treated.

As exemplified in FIG. 1, cellulosic feedstock 10 may be fed to reactor14 wherein cellulosic feedstock 10 is subjected to a first activationstep to produce a first activated cellulose stream 16. In the firstactivation step, cellulosic feedstock 10 may be treated at an elevatedtemperature and pressure to produce first activated cellulose stream 16comprising cellulose II and insoluble solids.

Reactor 14 may be a batch reactor or a continuous process reactor. Inthe case of a batch reactor, cellulosic feedstock 10 may be fed toreactor 14 and the reactor, which may be a stirred tank reactor, may beraised to the operating conditions for a desired time. If reactor 14 isa continuous flow reactor, then it may be a steam exposition reactor asis known in the art and may be maintained at the desired operatingcondition.

The first activation step may be conducted under conditions thatincrease the amount of cellulose II in the first activated cellulosestream relative to the amount of cellulose II in the feedstock.

The temperature may be greater than 190° C. and optionally greater than210° C., preferably greater than 220° C. and may be less than about 250°C. Accordingly, the process may be conducted at a temperature in therange of 190° C.-250° C., 210° C.-250° C., 220° C.-240° C., or 222°C.-230° C.

The pressure may be greater than 200 psig and optionally less than 500psig. Pressure in the reactor corresponds to temperature as persaturated steam thermodynamics as a minimum. In an embodiment, pressuremay be increased over and above that value by adding a pressurized gas,or adding superheat.

Cellulosic feedstock 10 may be subjected to the first activation stepfor less than 30 minutes, less than 20 minutes, less than 10 minutes orless than 5 minutes. The duration of the treatment time will varydepending upon many factors including severity of the activation step,e.g., the temperature and pressure of reactor 14.

It will be appreciated that the temperatures, pressures and duration oftreatment may be combined in any desired combination. Accordingly, forexample, the first activation step may comprise subjecting the feedstockto a pressure between 200 and 500 psig and a temperature between 200 and250° C. for 1 to 30 minutes, or a pressure between 200 and 500 psig anda temperature between 190 and 215° C. for less than 4 minutes.

Optionally, the first activation step is conducted in the presence ofwater. Water may be introduced into reactor 14 by one or more of beingpresent in cellulosic feedstock 10, being present in reactor 14 whencellulosic feedstock is introduced into reactor 14 and by beingintroduced by feed stream 12. The total amount of moisture that isintroduced into the reactor may be at least 30% and can be as high as90%. In a particular embodiment, there is 50% moisture going into thereactor.

The water which is present in reactor 14 may be in the form of steam orliquid water and is preferably in the form of liquid water. It will beappreciated that the temperature and pressure of the first activationstep may be selected such that liquid water is present in the reactor14.

First activated cellulose stream 16 may have a solids content of betweenabout 30% and 50% solids by weight. The solids will comprise chieflycellulose which may be subsequently subjected to a second activationstep. The solids may further comprise lignin, hemicellulose and minorcomponents such as ash, protein, or extractives.

Optionally, the cellulosic material may be subjected to one or morewashing steps, either under the same conditions or different conditions,after the first and/or second activation step. To this end, firstactivated cellulose stream 16 may be subjected to one or more washingsteps prior to the second activation step in order to remove solublenon-cellulosic components such as hemicellulose and some ash,extractives and lignin. The first wash removes these solubles and,because the solubles are at acidic pH, the wash step also reduces thealkali requirement in the second alkali activation stage.

As exemplified in FIG. 1, first activated cellulose stream 16 and washwater 20 may be introduced to wash reactor 18 to produce waste water 22and a washed first activated cellulose stream 24.

Wash water 20 may be hot water, such as water at a temperature betweenabout 40° C. and 100° C. or between about 50° C. and 95° C. Waste waterstream 22 may be treated and recycled in the process or elsewhere ordiscarded.

Wash reactor 18 may be any design known in the art. Optionally, washreactor 18 may be operated counter-currently and it may be acounter-current belt filter. Other filtration or separation methods maybe used such as a filter press, twin wire press, twin roll press, rotaryvacuum filter or a centrifuge.

As exemplified in FIG. 1, washed first activated cellulose stream 24 maybe fed to reactor 26 wherein washed first activated cellulose stream 24is subjected to a second activation step to produce a second activatedcellulose stream 30. In alternate embodiments, some or all of firstactivated cellulose stream may be introduced into reactor 26. Thefollowing description is based on FIG. 1 which exemplifies the use of afirst wash step. In the second activation step, washed first activatedcellulose stream 24 may be treated with an alkali at a lower temperaturethan the first activation step to produce a second activated cellulosestream comprising cellulose IV.

Reactor 26 may be batch reactor or a continuous process reactor. In thecase of a batch reactor, washed first activated cellulose stream 24 maybe fed to reactor 26 and the reactor, which may be a stirred tankreactor, may be raised to the operating conditions for a desired time.If reactor 26 is a continuous flow reactor, then it may be a steamexposition reactor as is known in the art and may be maintained at thedesired operating condition.

The second activation step may be conducted under conditions thatincrease the amount of cellulose IV in the second activated cellulosestream relative to the amount of cellulose IV in the washed firstactivated cellulose stream 24.

The temperature is optionally greater than 60° C., and may be less thanabout 180° C., less than about 160° C., less than 140° C., less thanabout 120° C., less than about 100° C. or less than about 80° C.Accordingly the process may be conducted at a temperature in the rangeof 60° C.-180° C., 60° C.-160° C., 60° C.-140° C., 60° C.-120° C., 60°C.-100° C. or 60° C.-80° C.

Optionally, the second activation step is conducted at superatmosphericpressure. For example, the superatmospheric pressure may be a pressurebetween about 0.1 and 400 psig.

Washed first activated cellulose stream 24 may be subjected to thesecond activation step for less than less than 180 minutes, less than120 minutes, less than 90 minutes or less than 60 minutes and optionallymore than 15 minutes, more than 30 minutes, or more than 45 minutes. Theduration of the treatment time will vary depending upon many factorsincluding severity of the activation step, e.g., the temperature andpressure of reactor 26.

It will be appreciated that the temperatures, pressures and duration oftreatment may be combined in any desired combination. Accordingly, forexample, the second activation step may comprise subjecting the firstactivated cellulose stream to a temperature between 60 and 240° C. for15 to 120 minutes at a pressure of 0 to 500 psig or a temperaturebetween 80 and 150° C. for at least 60 minutes at a pressure of 0 to 300psig.

As exemplified in FIG. 1, the second activation step preferablycomprises treating the first activated cellulose stream in the presenceof an alkali. The alkali may be introduced into reactor 26 in anymanner. For example, as exemplified, alkali stream 28 is introducedseparately into reactor 26. It will be appreciated that alkali stream 28may be introduced into reactor 26 prior to, concurrently with orsubsequent to the introduction of washed first activated cellulosestream 24 into reactor 26. Alternately alkali stream 28 may beintroduced into washed first activated cellulose stream 24 and acombined stream then introduced into reactor 26.

The alkali may comprise one or more of sodium hydroxide, potassiumhydroxide, magnesium hydroxide and ammonia. In one embodiment, thealkali is sodium hydroxide. In one embodiment, the alkali is loaded atbetween about 10% and 1%, between about 7% and 2% or preferably lessthan 6% of the total insoluble solids in first activated cellulosestream 24. The alkali swells cellulose and further breaks inter andintramolecular hydrogen bonds of the cellulose, thereby furthermodifying crystalline structure.

Optionally, the second activating step may be performed in the presenceof an oxidizing agent and/or an enzyme such as a laccase and/or a ligninmodifying enzyme.

Examples of oxidizing agents suitable for use in the second activationstep include, but are not limited to hydrogen peroxide (H₂O₂). In oneembodiment, the oxidizing agent is loaded at less than about 2% and/orgreater than about 0.0001% of the total insoluble solids in the firstactivated cellulose stream 16/24. In one embodiment, the oxidizing agentis loaded at less than about 1%, less than about 0.1% or less than about0.001% of the total insoluble solids in the first activated cellulosestream 16/24, optionally between about 1% and 0.0001%.

Examples of enzymes suitable for use in the second activation stepinclude, but are not limited to lignin modifying enzymes such as laccaseoxidizing enzyme.

Second activated cellulose stream 30 may have a solids content ofbetween about 5% and 50% solids by weight, preferably between about 20%and 35% solids. The solids will comprise chiefly cellulose which mayoptionally be recovered and recycled. Other components will behemicellulose and lignin, both less than 20%.

Optionally, second activated cellulose stream 30 may be subjected to oneor more washing steps after activation to remove alkali and solubilizedlignin.

Second activated cellulose stream 30 and wash water 34 may be introducedto wash reactor 32 to produce waste water 36 and a washed secondactivated cellulose stream 38. Wash reactor 32 may be operated in asimilar manner to wash reactor 18 or differently.

Wash water 34 may be hot water, such as water at a temperature betweenabout 50° C. and 95° C. or between about 60° C. and 95° C. Waste waterstream 36 may be treated and recycled in the process or elsewhere ordiscarded.

Wash reactor 32 may be any design known in the art. Optionally, washreactor 32 may be operated counter-currently and it may be acounter-current belt filter. Other filtration or separation methods maybe used, such as filter press, twin wire press, twin roll press, rotaryvacuum filter or centrifuge.

An advantage of subjecting a cellulosic feedstock to a first activationstep at a high temperature followed by a second activation step at alower temperature under alkali conditions has been shown to increase thelevel of glycan and decrease the level of lignin in second activatedcellulose stream 30 relative to a feedstock only subjected to a firsthigh temperature activation step. Without being limited by theory, it isbelieved that the two-step activation process described herein altersthe crystallinity of cellulose in the feedstock and improves thephysical and/or chemical characteristics of the cellulose for enzymatichydrolysis. In one embodiment, the two-step activation process resultsin activated cellulose comprising cellulose II and alkali-cellulose IV.

A skilled person will appreciate that cellulose exists in severaldifferent crystalline structures, corresponding to the location ofhydrogen bonds between and within strands. For example, naturallyoccurring cellulose found in cellulosic biomass is cellulose I, withstructures I_(α) and I_(β). Cellulose in regenerated cellulose fibers istypically cellulose II. Regenerated cellulose fibers refers to fibersproduced by the viscose process for viscose production of cellophane orrayon. The conversion of cellulose I to cellulose II is irreversible.The structures of cellulose III and cellulose IV may be produced throughvarious chemical treatments. The different crystalline forms ofcellulose can be identified by characteristic X-ray diffractionpatterns. Cellulose and the different crystalline structures ofcellulose are further described in Perez and Samain, “Structure andEngineering of Cellulose” Advances in Carbohydrate Chemistry andBiochemistry, Vol. 64, Elsevier (2010), which is hereby incorporated byreference in its entirety.

Washed second activated cellulose stream 38 may be subjected toenzymatic hydrolysis in enzymatic hydrolysis reactor 40 with one or morecellulase enzymes 42 to produce a glucose-rich sugar stream 44. It willbe appreciated that some or all of second activated cellulose stream 30may be subjected to enzymatic hydrolysis and accordingly only a part ornone of second activated cellulose stream 30 may be subjected towashing. The following description may apply to second activatedcellulose stream 30 whether or not subjected to a washing step.

It has surprisingly been determined that the activated cellulosecontaining cellulose II (which may be a combination of cellulose II andhydrated cellulose II) and cellulose IV (which may be alkali-celluloseIV) which may be produced by the two step activation process disclosedherein is particularly susceptible to enzymatic hydrolysis. Inparticular, the activated cellulose has shown a surprising ability toadsorb cellulase enzymes. Contacting the activated cellulose with one ormore cellulase enzymes in a glucose rich sugar stream may initiallyresult in the enzymes being adsorbed to the activated cellulose. Thecellulose may then be removed from the glucose-rich sugar stream andoptionally introduced into enzymatic hydrolysis reactor 40.

Therefore, the activated cellulose produced by any method disclosedherein may be subject to enzymatic hydrolysis to break the cellulosedown into cellulosic sugars such as glucose. Alternately the enzymatichydrolysis process disclosed herein may be used with any conventionalenzymatic hydrolysis cellulosic feedstock.

Accordingly, activated cellulose, optionally a second activatedcellulose stream as described herein may be contacted with one or morecellulase enzymes to produce the glucose-rich sugar stream. Asexemplified in FIG. 1, washed second activated cellulose stream 30 andenzyme stream 42 are introduced into enzymatic hydrolysis reactor 40 toproduce glucose rich sugar stream 44. Washed second activated cellulosestream 30 may be introduced into enzymatic hydrolysis reactor 40 priorto, concurrently with or subsequent to the introduction of enzyme stream42 into reactor 40. Alternately, or in addition, enzyme stream 42 may beintroduced into washed second activated cellulose stream 30 and thecombined stream may be introduced into enzymatic hydrolysis reactor 40.

Enzymatic hydrolysis reactor 40 may be any enzymatic hydrolysis reactorknown in the art and may operate on a batch or continuous basis.Enzymatic hydrolysis reactor 40 may operate at any conventionaltemperatures and pressures, cellulose loading, enzyme loading and thelike. For example, enzymatic hydrolysis reactor 40 may operate at atemperature range of 40° C. to 55° C.

The cellulase enzymes may be selected to break cellulose down intomonomeric sugars. For example, the cellulase enzymes may be selected tohydrolyze 1,4-beta-D-glycosidic linkages into monosaccharides. The oneor more cellulase enzymes may comprise an enzyme with at least one ofcellobiohydrolase, endoglucanase and beta-glucosidase activity. Whilecellulase enzyme preparations may be isolated from a number of sourcessuch as natural cultures of bacteria, yeast or fungi a person skilled inthe art will appreciate using enzymes produced using recombinanttechniques. Examples of commercially available enzymes suitable for usewith the methods described herein include, but are not limited to,Novozymes Ctec 2 or 3, AB Enzymes Rohament.

The one or more cellulase enzymes may be added at a loading of 0.1 to120 mg, 0.2 to 60 mg or 1 to 30 mg of enzyme protein per gram of glucan.In one embodiment, the cellulase enzymes are added at a loading of 0.1to 5 mg of enzyme protein per gram of glucan in the activated cellulose.In one embodiment, the one or more cellulase enzymes are added to theactivated cellulose at a loading of about 2 to about 60 Filter PaperUnits (FPU)/g of glucan, or optionally ata loading of about 2 to 30 or 1to 15 FPU/g of glucan. The one or more cellulase enzymes may be addedseparately to the activated cellulose directly or first combined with asurfactant and/or dispersant as described subsequently.

The one or more cellulase enzymes may be contacted with the activatedcellulose for a suitable length of time (e.g., for between 24-144 hours,between 48-144 hours, between 48-60 hours or between 24 and 72 hours) toin order to convert the cellulose into monomeric sugars throughenzymatic hydrolysis.

In some embodiments, at least about 70%, 75%, 80%, 85%, 90%, or 95% ofthe theoretical yield of glucose based on the glycan content of theactivated cellulose is converted to glucose during enzymatic hydrolysisto produce a glucose-rich sugar stream. In some embodiments, enzymatichydrolysis is conducted for a predetermined length of time or until apredetermined yield of glucose is obtained. After a certain amount oftime, the rate of glucose production from the enzymatic hydrolysis ofcellulose may decrease as the cellulose substrate is depleted or thepresence of glucose inhibits the activity of the cellulase enzymes.

Optionally, an activated cellulose may be contacted with one or morecellulase enzymes in the presence of a surfactant and/or a dispersant.In a preferred embodiment, the dispersant is polyaspartic acid.

It has surprisingly been determined that subjecting activated cellulose,particularly the activated cellulose disclosed herein, to enzymatichydrolysis in the presence of a surfactant and/or a dispersant such aspolyaspartic acid offers a number of advantages for the production ofmonomeric sugars. For example, the presence of the surfactant and/ordispersant may increase cellulase enzyme stability, help protect thecellulase enzymes from degradation, prevent irreversible binding and/orimprove cellulase enzyme activity. The presence of the surfactant and/ordispersant is also believed to improve the recovery of cellulase enzymesinto an enzyme recycle stream following enzymatic hydrolysis. Forexample, in some embodiments cellulase enzymes may be used for theenzymatic hydrolysis of activated cellulose, removed from the resultingglucose-rich sugar stream and recycled to enzymatic hydrolysis reactor40 or contacted with fresh activated cellulose for further enzymatichydrolysis. In some embodiments, cellulase enzymes may be used andrecycled in at least 3 or 4 rounds of enzymatic hydrolysis, each cyclelasting between 48 and 72 hours.

The surfactant may be a non-ionic surfactant, optionally a polysorbatesurfactant such as Tween. The surfactant may also be a blend ofsurfactants. In a preferred embodiment the surfactant is a blend ofTween 80, an alkoxylated glyceride and nonyl phenol. In one embodiment,the surfactant is present at a loading of less than about 2% and/orgreater than about 0.01%. In one embodiment, the surfactant is presentat a loading between 1% and 0.01%, between 0.5% and 0.05% or betweenabout 0.1% and 0.2% of the weight of the cellulose content in theactivated cellulose.

The dispersant may be an oligopeptide, optionally a non-enzymaticpolypeptide with molecular weight between 500 and 10,000 or between 1000and 5000. The oligopeptide may be polyaspartic acid. The polyasparticacid may have a molecular weight between 500 and 10,000, between 1000and 5000 or between 3500 and 4500. The polyaspartic acid may be presentat a loading of less than about 2% and/or greater than about 0.001% ofthe weight of the cellulose content in the activated cellulose. In someembodiments, the polyaspartic acid is present at a loading between 1%and 0.001%, between 0.25% and 0.025%, or about 0.1% of the weight of thecellulose content in the activated cellulose.

Optionally, the ratio of surfactant to dispersant (e.g., polyasparticacid) in the enzymatic hydrolysis mix is from 0.1:1 to 10:1, optionallyfrom 0.5:1 to 2:1.

Optionally, the molar ratio of dispersant (e.g., polyaspartic acid) tothe one or more cellulase enzymes is from 0.01 to 10:1.

Accordingly, an enzymatic hydrolysis mix comprising one or morecellulase enzymes, one or more surfactants and one or more dispersantsmay be used in any enzymatic hydrolysis process or may be used inassociation with any of the activation and enzymatic hydrolysisprocesses disclosed herein. The enzymatic hydrolysis mix is particularlysuitable for the enzymatic hydrolysis of activated cellulose comprisingcellulose II and cellulose IV as described herein.

The one or more cellulase enzymes, the surfactant and the dispersant maybe introduced singularly or in combinations or sub-combinations intoenzymatic hydrolysis reactor 40. For example, they may each be combinedseparately with the activated cellulose (e.g., each may be sequentiallyadded to stream 38 or stream 38 may be divided into 3 streams and thecellulase enzymes, the surfactant and the dispersant may each be addedto one of the divided out streams) prior to introducing stream 38 intoreactor 40. Alternatively, the one or more cellulase enzymes, thesurfactant and the dispersant may be combined together to form stream 42prior to combining the mixture with the activated cellulose (e.g.,introducing stream 42 into reactor 40 or introducing stream 42 intostream 38 prior to introducing stream 38 into reactor 40). Combining theenzymes, surfactant and the dispersant together prior to contacting theactivated cellulose is believed to facilitate the formation of a ternarycomplex that helps stabilize the enzyme and prevent enzyme degradation.Accordingly, the one or more cellulase enzymes may be combined with thesurfactant and the dispersant prior to subjecting the activatedcellulose to enzymatic hydrolysis. For example, the one or morecellulase enzymes may be combined with the surfactant and the dispersantfor at least 5 seconds, at least 10 seconds, at least 30 seconds or atleast 1 minute prior to contacting them with the activated cellulose(e.g., stream 38) or prior to subjecting the activated cellulose toenzymatic hydrolysis.

As exemplified in FIG. 1, glucose-rich sugar stream 44 may be subjectedto an enzyme removal step to obtain a glucose-rich and enzyme-poor sugarstream 48 and an enzyme recycle stream 50. The enzyme removal step maybe any enzyme removal step known in the art and may be conducted in anyequipment known in the art. Optionally, the enzyme removal stepcomprises contacting glucose-rich sugar stream 44, e.g., for a limitedtime, with cellulose, which may be an activated cellulose produced byany method disclosed herein.

For example, the enzyme removal step may comprise:

(a) contacting the glucose-rich sugar stream including enzymes withcellulose and obtaining cellulose with enzymes adsorbed thereon; and,

(b) subjecting the glucose-rich sugar stream to a cellulose removal stepto obtain the glucose-rich and enzyme poor sugar stream which has areduced level of cellulose and the enzyme recycle stream.

Optionally, step (a) comprises contacting the glucose-rich sugar streamwith activated cellulose, optionally a second activated cellulose streamproduced according to methods described herein.

Without being limited by theory, it is believed that enzymes in theglucose-rich sugar stream adsorb onto the cellulose such that removingthe cellulose from the glucose-rich sugar stream removes enzymes fromthe stream and results in glucose-rich and enzyme-poor sugar stream 48and enzyme recycle stream 50. In a particularly preferred embodiment,the cellulase enzymes in the glucose-rich sugar stream are in thepresence of a surfactant and a dispersant and the enzymes inglucose-rich sugar stream 44 are removed by contacting glucose richsugar stream 44 with activated cellulose 16, 24, 30, 38 produced usingthe methods described herein.

Accordingly a cellulose stream 52 may be introduced into reactor 46.Reactor 46 may comprise any reactor which may enable glucose-rich sugarstream 44 and cellulose to contact each other so as to withdraw enzymesfrom the solution and to separate the cellulose with the enzymesabsorbed thereon. Accordingly, for example, reactor 46 may comprise astirred tank reactor or a plug flow reactor for mixing the glucose-richsugar stream and cellulose to produce a mixed stream 54.

The glucose-rich sugar stream and cellulose may be contacted togetherfor less than about 2 hours, less than about 90 minutes or less thanabout 60 minutes and may be contacted together for between about 10minutes and 60 minutes, or between about 30 minutes and 90 minutes.

Subsequently, mixed stream 54 is subjected to a solid liquid separationstep is a separator 56. Separator 56 may be any separator known in thearts. Separator 56 may use any separation technique known in the artsuch a filtration, decantation, gravity separation, centrifugation, oruse of a press. For Example, separator 56 may comprise a filter, press,optionally a twin screw press, a twin wire press or a twin roll press.

Enzyme recycle stream 50 may be a high solids stream. For example,enzyme recycle stream may comprise greater than about 30%, greater thanabout 40% or greater than about 50% cellulosic solids.

Enzyme recycle stream 50 may be used for conducting enzymatic hydrolysison fresh activated cellulose. Alternately, enzyme recycle stream 50 maybe recycled to reactor 40. Accordingly, if reactor 40 is operated on abatch basis, a purge stream of glucose-rich sugar stream may bewithdrawn and treated to obtain recycle stream 50. Surprisingly, it hasbeen determined that enzymes recycled by this process generally maintaintheir activity after being recycled once, twice, three times or evenfour times. As a result, glucose-rich sugar stream 48 may comprisegreater than about 12% glucose, greater than about 14% glucose, greaterthan about 16% glucose or greater than about 18% glucose. Further, ayield of glucose that is greater than about 70%, greater than about 80%,greater than about 85%, greater than about 90% or greater than about 95%of a theoretical yield of glucose may be obtained.

Optionally, glucose-rich sugar stream 48 may comprises a detectablelevel of polyaspartic acid. In one embodiment, the sugar streamcomprises between about 1 ppb and 10000 ppm polyaspartic acid.

It has surprisingly been found that the glucose-rich sugar streamresulting from the enzymatic hydrolysis of the activated cellulosedisclosed herein contains about 5% non-glucose sugars which are one ormore of xylose, oligomers of xylose (xylo-oligosaccharide) and xylan.Xylo-oligosaccharides as described herein refer to polymers of xyloshaving a degree of polymerization (dp) of about 2 to about 10. Xylan asdescribed herein refers to polymers of xylose having a degree ofpolymerization (dp) of >10.

In a particular aspect the enzymatic hydrolysis of the activatedcellulose may be carried out using the hydrolysis mix as disclosedherein. In a further aspect the glucose rich sugar stream is preparedusing the cellulose activation method and/or the enzyme hydrolysismethod described herein.

Glucose obtained from sources such as corn using standard methods arealso known to have about 5% non-glucose sugars. However, the non-glucosesugars found in corn glucose are higher glycemic index sugars such asmaltose, maltotriose, higher saccharides of dextrose.

In one embodiment the non-glucose sugars found in the glucose rich sugarstream disclosed herein comprise about 1-8%, about 2-7% or about 3-6% ofthe dry matter of the composition and are one or more of xylose,xylo-oligosaccharides and xylan. In a particular embodiment the drymatter of the glucose-rich sugar stream was found to comprise 95%glucose, 4% xylose, 1% xylo-oligosaccharides.

Glucose obtained from the methods disclosed herein can be converted tofructose using known methods such as glucose isomerization to fructoseas described, for example, by S. Z. Dziedzic et al., “Handbook of starchhydrolysis products and their derivatives” Dec. 31, 1995, pages 55-58,which is hereby incorporated herein by reference. It has been found thatthe fructose obtain from conversion of the glucose of obtained from themethods disclosed herein also contains about 3-5% non-glucose sugarwherein the non-glucose sugar is xylose and/or oligomers of xylose.

In accordance with a further aspect of the invention there is provided afructose-rich sugar stream prepared by conversion of the glucose in theglucose-rich sugar stream disclosed herein to fructose. Thefructose-rich sugar stream also comprises non-fructose sugar, whereinthe non-fructose sugar is one or more of xylose, xylo-oligosaccharideand xylan. In one embodiment the non-fructose sugars comprise about1-8%, about 2-7% or about 3-6% of the dry matter of the composition.

In accordance with another aspect of the invention there is provided alower glycemic index glucose product or lower glycemic index fructoseproduct wherein the glucose product or fructose product comprises about1-8%, about 2-7% or about 3-6% preferably 5% non-glucose or non-fructosesugar and wherein the non-glucose or non-fructose sugar is one or moreof xylose, xylo-oligosaccharides and xylan.

In a further aspect the glycemic index of the glucose product orfructose product is lower than the glycemic index of conventionalglucose or fructose syrup produced in a conventional manner. Theglycemic index (GI) can be measured using methods known in the art forexample as described in “In vitro method for predicting glycemic indexof foods using simulated digestion and an artificial neural network” R.L. Magaletta et al., Cereal Chemistry vol. 87, no. 4, 2010.

The glucose product or fructose product obtained by the methodsdescribed herein can be substituted for higher glycemic index glucose orfructose in production of various foods and drinks to provide a lowerglycemic index product. Lower glycemic index foods and drinks mayprovide health advantages in the management of blood sugar and insulinlevels which may in turn reduce the risk of heart disease and/ordiabetes. Foods having a lower glycemic index may also be useful incontrolling appetite and weight loss.

It will be appreciated that one or more of the embodiments describedherein for the activation of a cellulosic feedstock may be used togetherwith one or more embodiments described herein for the enzymatichydrolysis of cellulose in order to produce cellulosic sugars from acellulosic feedstock.

What has been described above has been intended to be illustrative ofthe invention and non-limiting and it will be understood by personsskilled in the art that other variants and modifications may be madewithout departing from the scope of the invention as defined in theclaims appended hereto. The scope of the claims should not be limited bythe preferred embodiments and examples, but should be given the broadestinterpretation consistent with the description as a whole.

While the above disclosure generally describes the present application,a more complete understanding can be obtained by reference to thefollowing specific examples. These examples are described solely for thepurpose of illustration and are not intended to limit the scope of thedisclosure. Changes in form and substitution of equivalents arecontemplated as circumstances might suggest or render expedient.Although specific terms have been employed herein, such terms areintended in a descriptive sense and not for purposes of limitation.

The following non-limiting examples are illustrative of the presentdisclosure:

EXAMPLES Example 1 Treatment of Sugarcane Bagasse for ActivatingCellulose

Various methods for activating cellulose were investigated usingsugarcane bagasse. Sugarcane bagasse was subjected to either a firststeam treatment step 220° C., 5 minutes residence time followed by a hotwater wash using water at 80° C. or a first steam treatment followed bytreatment with alkaline hydrogen peroxide. Alkali wash at 90° C., 60minutes or 120 minutes, and 1% peroxide loading on solids.

As shown in Table 1, the use of the two-step treatment with a steamtreatment step followed by alkaline hydrogen peroxide significantlyincreased the level of glucan and decreased the amount of ligninrelative to treatment with steam and a hot water wash.

TABLE 1 Influence of alkaline peroxide treatment on the chemicalcomposition of the water insoluble component of steam pretreated andsubsequently hot water washed sugarcane bagasse* (% dry weight). AHP-60and AHP-120 refer to 60 minute and 120 minute treatments. Steampretreated and hot water washed AHP - 60* AHP - 120** Arabinan   BDL****BDL BDL Galactan BDL BDL BDL Glucan 48.3 (0.7) 79.3 (1.4) 78.2 (0.7)Xylan 3.6 (0.1) 3.7 (0.1) 3.7 (0.1) Mannan BDL BDL BDL Lignin (Acid 41.6(1.6) 12.9 (0.3) 12.9 (0.3) insoluble)** Acid soluble lignin 0.7 (0.0)0.6 (0.0) 0.5 (0.0) Ash 3.2 (0.4) 1.7 (0.4) 1.9 (0.7) *Alkaline hydrogenperoxide treatment **Alkaline hydrogen peroxide treatment ***A minorfraction of the lignin may contain ash components ****Below detectablelevel

Example 2 Enzymatic Hydrolysis of Activated Sugarcane Bagasse

The water insoluble cellulosic components prepared in Example 1 werethen subjected to enzymatic hydrolysis for 72 hours. Comet additiveS-001, comprising a mixture of a surfactant Tween 80 and a dispersantpolyaspartic acid with a MW of 3500-4500 in a 1:1 ratio was also addedto the enzymatic hydrolysis mix for the alkali treated sugar canebagasse.

As shown in Table 2, enzymatic hydrolysis of alkali treated sugarbagasse in the presence of the Comet additive S-001 resulted in aglucose yield of 105.1 grams of glucose per gram of glucan, approachingthe theoretical yield of ˜110 grams of glucose per gram of glucan.

TABLE 2 Monomeric glucose yield after the 72 hour enzymatic hydrolysisof the water insoluble cellulosic component of steam pretreated andsubsequently alkaline peroxide treated sugarcane bagasse (expressed as gper 100 g glucan**). Substrate Glucose yield Hot water washed sugar canebagasse 79.1 (2.6)*** Alkali treated sugar cane bagasse + Comet 105.1(1.5) additive-S-001 * Cellulase loading: 31 mg protein per g of glucan**100 g glucan should theoretically release ~110 g glucose. ***Values inthe bracket represent standard deviations of triplicates

Furthermore, as shown in Table 3, enzymatic hydrolysis in the presenceof Comet additive S-001 did not alter the yield of monomeric xylosecompared to alkali treated sugarcane bagasse subject to enzymatichydrolysis without the additive. Accordingly, the additive did notadversely affect the yield.

TABLE 3 Monomeric xylose yield after the 72 hour enzymatic hydrolysis*of the water insoluble cellulosic component of steam pretreated andsubsequently alkaline peroxide treated sugarcane bagasse (expressed as gper 100 g substrate**). Substrate Xylose yield Alkali treated sugarcanebagasse 2.7 (0.2)*** Alkali treated sugarcane bagasse + 2.7 (0.1) Cometadditive-S-001 *Refer to Table 2 for enzymatic hydrolysis conditions**100 g substrate should theoretically release 4.1 g xylose (refer toTable 1 for xylan content). ***Values in the bracket represent standarddeviations of triplicates

Analysis of the fraction of the total protein content present in thesupernatant after 72 hours of enzymatic hydrolysis is shown in Table 4.The use of Comet additive S-001 resulted in a higher fraction of totalprotein, indicative of the higher levels of enzymes within thesupernatant and improved enzyme stability.

TABLE 4 Fraction of the total protein present in the supernatant after72 hours (expressed as g per 100 g protein added**). Fraction of thetotal proteins in the liquid Hot water washed sugar cane bagasse —Alkali treated sugarcane bagasse 61.2 (2.2) Alkali treated sugarcanebagasse + 77.2 (1.1)** Comet additive-S001 **Values in the bracketrepresent standard deviations of triplicates

The alkali treated sugarcane bagasse was subject to multiple rounds ofrecycle hydrolysis in the presence of Comet additive S-001. As shown inTable 5, recycle hydrolysis was able to produce a high yield and a highconcentration of glucose with minimal loss of enzymes over repeatedrounds of enzymatic hydrolysis.

TABLE 5 Results of the recycle hydrolysis (Total 16% glucan)* of thewater insoluble cellulosic component of steam pretreated andsubsequently alkaline peroxide treated sugarcane bagasse. HydrolysisGlucose Dissolved Fraction of the interval and Glucose concentrationXylose solids proteins in the substrate addition yield (%) (% wt/vol.)yield*** (wt/wt) supernatant***** After 48 83.9 (1.4)**** 16.1 (0.3) 1.9(0.2) 19.1 (0.2) 58.4 (1.3) hours** After next 48 83.1 (0.9) 15.9 (0.2)2.2 (0.0) 18.8 (0.2) 60.1 (2.4) hours After next 48 90.9 (0.3) 18.2(0.1) 2.5 (0.0) 20.9 (0.1) 64.2 (0.8) hours After next 48 92.3 (2.1)18.1 (0.4) 2.3 (0.1) 20.7 (0.5) 67.7 (1.5) hours *16% glucan loading(~20% solids loading) of the alkali treated substrate. The reaction wasconducted at a total 20 L scale. **Cellulase loading: 31 mg protein perg of glucan added in the beginning of the hydrolysis. 0.2% S-001 in thereaction mixture, 6 mg supplement enzyme protein per recycle***expressed as g/100 g substrate. 100 g substrate should theoreticallyrelease 4.1 g xylose (refer to Table 1 for xylan content). ****Values inthe bracket represent standard deviations of triplicates *****Does notaccount for enzyme adsorbed onto substrate

Example 3 Treatment of Wheat Straw for Activating Cellulose

Methods for activating a cellulose feedstock were investigated usingwheat straw. Wheat straw was subjected to a first steam treatment stepat 220° C., 5 minute residence time, followed by a hot water wash at 80°C., followed by alkaline wash at 90° C. for 60 minutes and 1% peroxideloading on solids.

As shown in Table 6, the use of the two-step treatment with alkalinehydrogen peroxide resulted in water insoluble components with a highlevel of glucan (75.1%).

TABLE 6 Chemical composition of the water insoluble component of steampretreated and subsequently alkaline peroxide treated wheat straw* (%Dry weight) Arabinan   BDL*** Galactan BDL Glucan 75.1 (0.6) Xylan 8.1(0.1) Mannan BDL Lignin (Acid insoluble)** 13.5 (0.4) Acid solublelignin 0.4 (0.0) Ash 1.2 (0.3) *Solids yield after the peroxidetreatment was 75.7. Alkaline peroxide treatment was conducted at 10%consistency, pH 11.5 and 1% peroxide solution, 80° C. for 2 hours. **Aminor fraction of the lignin may contain ash components ***Belowdetectable level

Example 4 Enzymatic Hydrolysis of Activated Wheat Straw

The water insoluble cellulosic components of steam treated wheat strawor steam treated and subsequently alkali treated wheat straw weresubject to enzymatic hydrolysis as shown in Tables 7-9. The surfactantTween 80 was also added, as noted in the table.

TABLE 7 Monomeric glucose yield during the enzymatic hydrolysis (10%glucan loading)* of the water insoluble cellulosic component of steampretreated and subsequently alkaline peroxide treated wheat straw(expressed as g per 100 g glucan**). 24 hours 72 hours Hot water washedwheat straw —**** 85.0 (2.1) Hot water washed wheat straw + —   89.1(0.5) Tween 80*** Peroxide treated wheat straw 83.7 (1.1)***** 97.0(0.3) Peroxide treated wheat straw + 90.4 (0.1) 102.3 (2.9) Tween 80*13.3% solids loading for peroxide treated substrate & 17.9% solidsloading for steam pretreated wheat straw in order to obtain 10% glucanloading. Cellulase loading: 31 mg protein (CTec 2) per g of glucan **100g glucan should theoretically release ~110 g glucose. ***0.2% Tween 80in the reaction mixture ****was not liquefied enough to obtain arepresentative sample for analysis *****Values in the bracket representstandard deviations

TABLE 8 Monomeric xylose yield after the 72 hour enzymatic hydrolysis***(10% glucan loading)* of the water insoluble cellulosic component ofsteam pretreated and subsequently alkaline peroxide treated wheat straw(expressed as g per 100 g substrate**) Xylose yield Hot water washedwheat straw 6.1 (0.0) Hot water washed wheat straw + Tween 6.3 (0.2) 80Alkaline peroxide treated wheat straw 5.8 (0.0) Alkaline peroxidetreated wheat straw + 6.0 (0.1) Tween 80

TABLE 9 Dissolved solids present in the 72 hour enzymatic hydrolysate ofsteam pretreated and subsequently alkaline peroxide treated wheat straw(% wt/wt) Dissolved Solids (% wt/wt) Hot water washed wheat straw 13.8(0.1) Hot water washed wheat straw + Tween 12.3 (0.0) 80 Alkalineperoxide treated wheat straw 14.1 (0.2) Alkaline peroxide treated wheatstraw + 13.9 (0.1) Tween 80

Further investigations of the enzymatic hydrolysis of steam treated andsubsequently alkaline peroxide treated wheat straw with the addition ofa surfactant (Tween 80) were performed as set out in Tables 10-12.

TABLE 10 Monomeric glucose yield during the fed-batch enzymatichydrolysis (10% glucan loading)* of the water insoluble cellulosiccomponent of steam pretreated and subsequently alkaline peroxide treatedwheat straw with the addition of Tween 80 (expressed as g per 100 gtotal glucan**). After first After next After next After next 36 Hours24 hours 24 Hours 24 Hours Glucose 93.4 (0.5)*** 72.7 (1.3) 61.5 (0.7)49.7 (2.8) yield** Dissolved 12.9 (0.1) 10.0 (0.1) 8.4 (0.0) 6.9 (0.3)solids *13.3% solids loading for peroxide treated substrate to obtain10% glucan loading. 0.2% Tween 80 in the reaction mixture only in thebeginning. Cellulase loading: 31 mg protein (CTec 2) per g of glucan and3.1 mg/g glucan before adding every fresh batch of the substrates. **100g glucan should theoretically release ~110 g glucose. ****Values in thebracket represent standard deviations

TABLE 11 Monomeric xylose yield during the fed-batch enzymatichydrolysis (10% glucan loading) of the water insoluble cellulosiccomponent of steam pretreated and subsequently alkaline peroxide treatedwheat straw expressed as g per 100 g total substrate used for hydrolysisin every stage). After first 36 hours of enzymatic hydrolysis 5.5 (0.1)After next 24 hours 4.3 (0.0) After next 24 hours 3.6 (0.1) After next24 hours 2.9 (0.0)

TABLE 12 Fraction of the total protein present in the supernatant priorto the addition of every batch of fresh substrates and enzymes(expressed as % of the total protein added**). Batch Fed batchhydrolysis* hydrolysis** After first After the next After the next Afterthe next At the end 36 hours 24 hours 24 hours 24 hours of 72 hoursFraction of 61.6 (1.1) 63.3 (0.5) 63.1 (1.7) 66.2 (2.4) 72.7 (2.8) thetotal proteins in the liquid *31.1 mg protein loading/g cellulose in thebeginning of first batch of hydrolysis and 3.1 mg protein/g cellulosebefore the addition of every fresh batch of substrates. 10% glucanconsistency in the first 36 hours followed by the addition of 10% glucanin every subsequent 24 hours. **Protein loading: 31.1 mg/g cellulose &10% glucan consistency

Example 5

A cellulosic glucose product produced by the activation and enzymaticconversion methods described herein was prepared and was determined tohave the following specifications:

Chemical and Physical Data

-   Total solids 50-70%-   Moisture 30-50%

Composition (Dry Matter Basis):

Glucose 95%  Xylose 4% Xylo-oligosaccharides 1% Ash <0.01%   

-   pH: 3-5-   Conductivity: (30% DS) 50 ps/cm-   Specific gravity: 1.2-   Appearance: clear solution-   Odor: sweet

Mineral Ash Content (PPM

Chloride 16 Sulphate <1 Calcium 5 Potassium <1 Magnesium <1 Sodium 2Phosphorous 2

Glycemic Index Data

The cellulosic glucose product having the composition described abovewas found to have a glycemic index (GI) of 72. By comparison glucosealone is known to have a glycemic index of 100. Dextrose is also knownto have a glycemic index of 100 while maltose and maltodextrin are knownto have glycemic indexes of 105 and 110 respectively.

The present disclosure has been described with reference to what arepresently considered to be the examples, it is to be understood that thedisclosure is not limited to the disclosed examples. To the contrary,the disclosure is intended to cover various modifications and equivalentarrangements included within the spirit and scope of the appendedclaims.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

1. A two-step method for activating a cellulosic feedstock, the methodcomprising: (a) subjecting the feedstock to a first activation stepwherein the feedstock is treated at a temperature greater than 190° C.and a pressure greater than 200 psig to produce a first activatedcellulose stream comprising cellulose II and insoluble solids; (b)subjecting the first activated cellulose stream to a second activationstep wherein the first activated cellulose stream is treated with analkali at a lower temperature than the first activation step to producea second activated cellulose stream comprising cellulose IV.